The term “mass” is used below to refer to a “charge-related mass” m/z, which is a physical measure, measured by any type of mass spectrometry. The term “mass” therefore does not refer to a “physical mass” m unless clearly indicated otherwise. The dimensionless number z represents a number of excess elementary charges on an ion; e.g., the number of electrons or protons of the ion that are effective externally as ion charge. Thus, the charge-related mass m/z is a mass fraction per elementary ion charge. The terms “light ions” and “heavy ions” are used below to respectively describe ions with low and high charge-related masses m/z. The terms “mass spectrum” and “mass discrimination” relate to the charge-related masses m/z. The terms “dalton” or “Da” describe a mass unit as well as a charge-related mass unit because a dalton is non-coherently assigned to the officially adopted International System of units (SI).
A time-of-flight mass spectrometer in which a primary ion beam undergoes pulsed acceleration at right angles to the original direction of flight of ions is referred to as an orthogonal time-of-flight mass spectrometer (OTOF-MS). FIG. 1 schematically illustrates a simplified embodiment of such an OTOF mass spectrometer 100. An ion pulser 12 is included in a mass analyzer of the mass spectrometer 100 at a first end of the flight path 13. The mass analyzer accelerates a section of the primary ion beam 11, for example a string-shaped ion packet, into the flight path 13 at right angles to the previous direction of the beam 11. This process provides a ribbon-shaped secondary ion beam 14 that includes individual, transverse, and string-shaped ion packets. Each of the string-shaped ion packets includes ions of equal mass. The string-shaped ion packets that include light ions fly relatively quickly, whereas the string-shaped ion packets that include heavier ions fly relatively slowly. The direction of flight of the ribbon-shaped secondary ion beam 14 lies between the previous direction of the primary ion beam 11 and the direction of acceleration at right angles because the ions retain speed in the original direction of the primary ion beam 11. The time-of-flight mass spectrometer 100 preferably includes a velocity-focusing reflector 15 to reflect the whole width of the ribbon-shaped secondary ion beam 14 with the string-shaped ion packets, focuses velocity spread of the beam 14, and directs beam 14 towards a flat detector 16.
The ion pulser 12 may operate with repetition frequencies between five and thirty kilohertz (kHz). Thus, between 5,000 and 30,000 individual spectra per second may be acquired, and summed in real time over a predetermined time span between about one twentieth of a second and twenty seconds to form a sum spectrum. This provides the sum spectra with a high dynamic measurement range, even where relatively few ions are measured in each individual spectrum. To scan substance peaks that separate in liquid chromatographs or capillary electrophoresis devices, the individual spectra are typically summed over a time span of one second to form a sum spectrum.
Today, time-of-flight mass spectrometers with orthogonal ion acceleration typically no longer use a continuous ion beam with ions flowing without interruption into the ion pulser. Rather, the ions are typically first collected in an ion storage device to increase the mass spectrometer sensitivity. U.S. Pat. No. 5,689,111 discloses such an ion storage device for an OTOF-MS.
The ion storage device 7 of FIG. 1 is an RF multipole rod system (e.g., a quadrupole rod system) terminated at its ends with aperture lenses 6 and 9. The ion storage device 7 is surrounded by an insulating casing that is filled with collision gas by the gas feeder 8 such that the ions in the interior thereof practically come to rest after a short damping period. The ions are extracted from the ion storage device 7 with low kinetic energy by switching a potential at the extraction lens 9, which directs the ions as a fine ion beam 11 to the ion pulser 12. The ions of the ion beam 11 fly through the pulser 12 with a uniform, relatively low kinetic energy of between about 15 and 20 electron volts. The relatively slow-flying ions are pulsed out of the ion pulser 12, using high acceleration voltages perpendicular to the previous direction of flight of the ions, into the flight path 13 of the time-of-flight mass spectrometer 100.
As the ions are extracted from the ion storage device 7 and transferred into the ion pulser 12, some mass separation takes place because, on the one hand, the light ions fly faster at an equal kinetic energy while, on the other hand, the light ions may be extracted faster from the ion storage device 7. The light ions therefore arrive at the ion pulser 12 first, and their density inside the pulser 12 decreases dramatically because the main bunch of the light ions extracted first from the storage device 7 has already left the ion pulser 12. The heavier ions reach the ion pulser 12 once the number of light ions in the ion pulser 12 has already greatly decreased. The number of heavier ions in the ion pulser 12 also passes through a relative maximum and then decreases again. The composition of the ions in the ion pulser 12 regularly changes until the pulsed ejection.
The time at which the pulsed ejection takes place determines the composition of the ions in the individual spectrum measured, which results in mass discrimination. Typically, as the number of ions of a specific mass in the ion pulser 12 increases, the lower the kinetic energy of the ions becomes because the ions fly more slowly, which increases the mass discrimination. Part of the mass discrimination occurs because the path between the multipole ion storage device 7 and the ion pulser 12 is not arbitrarily short for a variety of reasons; another part of the mass discrimination is generated while the ions are being extracted from the ion storage device. The ion storage device 7 is usually closed again shortly before the ion pulser 12 ejects the next pulse.
The ion storage device 7 includes collision gas for the purpose of collision focusing and to damp ion motion as effectively as possible. The ions thus collect in a relatively motionless state in the axis of the ion storage device 7. The ions therefore may be taken from the ion storage device 7 relatively easily and with relatively little energy spread. The ion pulser 12, in contrast, is configured in a region with a relatively strong vacuum to prevent the ions from colliding with residual gas molecules. The ions therefore typically pass through several differential pumping stages between the ion storage device 7 and the ion pulser 12. FIG. 1 illustrates, for example, an arrangement of einzel lens 10 mounted into a wall between two pump stages. The transfer of the ions from the annular extraction aperture 9 of the ion storage device 7 to the ion pulser 12 takes place in the ion beam 11 by free flight with relatively little collisions. The distance between the extraction lens 9 and a center of the ion pulser 12 may be between about five to eight centimeters.
The formation of the fine ion beam 11 is particularly important for the mass resolving power of the time-of-flight analyzer. The ion beam 11 should be a parallel beam of small diameter with slow ions of uniformly low energy (e.g., around fifteen electron volts). The fine ion beam 11 is formed where ion motion in the ion storage device 7 is effectively damped and electrical perturbations that may affect the quality of the ion beam 11 are reduced (e.g., minimized). Such electrical perturbations may be caused by switching the potential of the extraction lens 9, or residual fringe fields of the RF voltage at the ion storage device 7.
An example of mass discrimination between different ionic species as described above is graphically illustrated in FIG. 2 as a function of time. The curves in FIG. 2 are derived from measurements of the spectra of a mixture of substances whose masses range from m=78 Da to m=2722 Da, where singly charged ions (z=0) were evaluated. The spectra was measured, for example, for ionic species with 78, 118, 322, 622, 922, 1222, 1522, 1822, 2122, 2422 and 2722 dalton. The mass spectra were acquired with different time delays between the opening of the extraction lens 9 and the pulsing of the ion pulser 12; e.g., the time delays ranged from about 8 to 190 microseconds. From the spectra, the characteristics of the intensities of the different ionic species were generated as a function of the delay time and are normalized to the maximum in each case. The measurements show that a mass spectrum which is acquired with a delay time of 10 microseconds only measures ions of the mass m=78 u; i.e., there are no ions with higher masses in the mass spectrum. If one acquires a mass spectrum with a longer delay time, however, some ions with lower masses are contained, but only with low intensity. For example, with a delay of around 160 microseconds, the ions of all masses may be measured simultaneously, but not all with maximum sensitivity; e.g., the light ions have already dropped to around five percent of their maximum value.
Time-of-flight mass spectrometers of the type of FIG. 1 may be used in protein analysis. In peptide or protein analyses with electrospray ionization, multiply charged ions may be produced; e.g., the ions of the charge level with the largest number of ions may usually be found in the range of 900 Da<m/z<1500 Da, even if the proteins have a high mass m of several thousand daltons. The ions of proteins of high mass appear predominantly multiply charged, the charge level with the maximum intensity regularly being in the range of 900 Da<m/z<1500 Da. Peptides and proteins therefore may be optimally acquired with a delay time of 100 microseconds because the ions are measured here with more than eighty percent of the maximum intensity that may be achieved by varying the delay time. Thus, these instruments are ideal for this type of analysis. However, if ions with masses m/z>2700 Da are present in the mixture of ions to be analyzed, the ions do not appear in the mass spectrum. Furthermore, the ions of mass m/z=100 Da appear with between five and ten percent of the intensity which they would have with a mass-specific optimum setting.
Adaptation of the delay time is disclosed in U.S. Pat. Nos. 6,507,019 and 6,689,111.
Focusing on one mass range may be disadvantageous for many types of analysis such as, for example, quantitative analyses of protein mixtures where interesting proteins were labeled with “reporter groups”. The reporter groups are split off as singly charged ions during the ionization and are used for quantitative measurement. The reporter groups often have masses between m=90 Da and m=120 Da. If the delay time is set to 100 microseconds, the ions of the reporter groups appear with between five and ten percent of their maximum intensity. Since the proteins to be measured quantitatively (and with them the reporter groups) usually occur in low concentrations, they may not be evaluated effectively in the analyses in the vast majority of cases. Methods are therefore being sought in which peptide and protein ions as well as the ions of the reporter groups may be measured with high sensitivity.
Various methods and devices have been developed in an effort to reduce or eliminate mass discrimination. U.S. Pat. No. 6,794,604, for example, discloses a “mass selective ion trap” that first releases heavier ions, and subsequently releases increasingly lighter and lighter ions, where the release times are adjusted so that all the ions reach the ion pulser at the same time. In contrast, E.P. Publication No. 1 315 195, which makes no attempt to eliminate mass discrimination, discloses ions with m/z ratios within a first range are transferred from a selective ion trap to the OTOF for a first individual spectrum, while ions with m/z ratios outside this first range are essentially not transferred to the OTOF. Ions with m/z ratios within a second range are subsequently transferred from a selective ion trap to the OTOF for a second individual spectrum, while ions with m/z ratios outside the second range are essentially not transferred to the OTOF. Thus, in both the ‘604 patent and the ‘195 Publication, the term “mass-selective ion trap” is used to describe an ion trap that may mass-selectively eject the ions. Attempts at a mass selective trap fail, however, because the trap unavoidably ejects the ions with a high energy spread of a few tens to a few hundred electron volts because the ions are accelerated with different strengths upon exiting the trap, depending on the randomly prevailing phase of the internal RF field.
U.S. Pat. No. 7,582,864 discloses an RF quadrupole rod system, at the end of which a blocking pseudopotential is created at the exit aperture by a non-balanced RF voltage. By gradually reducing the pseudopotential barrier, first heavy, then lighter and lighter ions are allowed to exit the RF quadrupole rod system. While the damaging influence of the RF field on the exiting ions is smaller, it still interferes enough that the highest possible mass resolution is no longer achieved in the OTOF-MS.
There is a need for a method with which an OTOF-MS may be operated in such a way that, as the sum spectra are being acquired, the ions of several mass ranges of interest may be measured without significant losses; e.g., with relatively high ion yield and with relatively high mass accuracy.